Alberto Isla

Brain tumor and pregnancy

Objective To establish the indications for surgical intervention, at the appropriate gestational duration, for brain tumors in pregnant women, and to evaluate any association pregnancy hormones may have with the rate of growth or development of complications of brain tumors. Methods We observed seven women with brain tumors associated with pregnancy in a series of 126,413 pregnancies from 1983 to 1995. One woman presented with symptoms of intracranial hypertension, and neurologic signs were the first symptoms in two other women. One woman presented with a sudden hemorrhagic lesion, and three had focal seizures. All were evaluated with computed tomography, magnetic resonance imaging, or both. Results Six of the seven women had surgery; the seventh was treated with radiotherapy because the neuroradiologic studies suggested a quickly growing brainstem glioma. Diagnoses were confirmed on histology in six women: two with meningiomas, two with ependymomas, and two with low-grade astrocytomas (one of these had multiple astrocytomas). Estrogen and progesterone receptors were studied in two cases (one meningioma and one astrocytoma) and were present in both. Conclusion Management of brain tumors should be tailored to the individual patient. There may be a relation between pregnancy hormones and the rate of brain tumor growth mediated through specific intracellular receptors.

Genetic and epigenetic alteration of the NF2 gene in sporadic meningiomas

The role of the NF2 gene in the development of meningiomas has recently been documented; inactivating mutations plus allelic loss at 22q, the site of this gene (at 22q12), have been identified in both sporadic and neurofibromatosis type 2–associated tumors. Although epigenetic inactivation through aberrant CpG island methylation of the NF2 5′ flanking region has been documented in schwannoma (another NF2‐associated neoplasm), data on participation of this epigenetic modification in meningiomas are not yet widely available. Using methylation‐specific PCR (MSP) plus sequencing, we assessed the presence of aberrant promoter NF2 methylation in a series of 88 meningiomas (61 grade I, 24 grade II, and 3 grade III), in which the allelic constitution at 22q and the NF2 mutational status also were determined by RFLP/microsatellite and PCR‐SSCP analyses. Chromosome 22 allelic loss, NF2 gene mutation, and aberrant NF2 promoter methylation were detected in 49%, 24%, and 26% of cases, respectively. Aberrant NF2 methylation with loss of heterozygosity (LOH) at 22q was found in five cases, and aberrant methylation with NF2 mutation in another; LOH 22q and the mutation were found in 16 samples. The aberrant methylation of the NF2 gene also was the sole alteration in 15 samples, most of which were from grade I tumors. These results indicate that aberrant NF2 hypermethylation may participate in the development of a significant proportion of sporadic meningiomas, primarily those of grade I. ©2005 Wiley‐Liss, Inc.

CpG island methylation status and mutation analysis of the RB1 gene essential promoter region and protein-binding pocket domain in nervous system tumours

A series of 136 nervous system tumours were studied to determine the methylation status of the CpG island contained within the promoter region of the RB1 gene, as well as mutation analysis of the essential promoter region and exons 20–24 (and surrounding intronic regions) coding for the protein-binding pocket domain. Methylation of the RB1 CpG island was detected in 26 samples corresponding to nine glioblastomas, three anaplastic astrocytomas, one mixed oligo-astrocytoma, one ependymoma, two medulloblastomas, two primary central nervous system lymphomas, two neurofibrosarcomas, and six brain metastasis from solid tumours. No inactivating mutations were found within the RB1 promoter region, whereas one glioblastoma and one oligodendroglioma displayed similar sequence variations consisting of 12 and 8 base pair deletions at intron 21. These results suggest that RB1 CpG island hypermethylation is a common epigenetic event that is associated with the development of malignant nervous system tumours.

DNA methylation of multiple promoter-associated CpG islands in meningiomas: relationship with the allelic status at 1p and 22q

The purpose of this research was to examine the DNA methylation profile of meningiomas. Accordingly, we examined the DNA methylation status of ten tumor-related genes (RB1, p16INK4a, p73, MGMT, ER, DAPK, TIMP-3, p14ARF, THBS1, and Caspase-8) in 98 meningiomas (68 grade I; 27 grade II; and 3 grade III samples) using methylation-specific PCR and sequencing. The most frequently methylated genes were THBS1 (30%), TIMP-3 (24%), p16INK4a (17%), MGMT (16%), p73 (15%), ER (15%), and p14ARF (13%), whereas methylation was relatively rare in the other genes (<10%). Methylation occurred in at least one gene in 77.5% of the cases and in three or more genes in 25.5%. Methylation was tumor specific since it was absent in the controls: two non-neoplastic meningeal samples and two non-neoplastic brain samples. The frequency of aberrant gene methylation in grade I versus grade II–III tumors showed some differences for TIMP-3, THBS1, MGMT, p16INK4a and p73; these differences reached statistical significance for TIMP-3: 18% in grade I versus 40% in grade II–III (P<0.02). Our previous loss of heterozygosity studies provided the allelic constitution at 1p and 22q for 60 of the 98 meningiomas included in this report. The level of aberrant promoter methylation increased in tumors (30 samples) displaying 1p loss (either isolated or as concurrent deletion at 1p/22q; P=0.014). These meningiomas primarily accumulated the epigenetic changes of THBS1 (14/30; 47%; P<0.005), TIMP-3 (12/30; 40%; P<0.05), p73 (10/30; 26%; P<0.02) and p14ARF/p16INK4a(7/30 each one; 23%; not significant). Our findings indicate that aberrant DNA methylation of promoter-associated CpG islands in meningiomas contributes to the development of these tumors.

Mutation analysis of the p73 gene in nonastrocytic brain tumours

Loss of heterozygosity (LOH) involving the distal chromosome 1 p36 region occurs frequently in nonastrocytic brain tumours, but the tumour suppressor gene targeted by this deletion is unknown. p73 is a novel gene that has high sequence homology and similar gene structure to the p53 gene; it has been mapped to 1 p36, and may thus represent a candidate for this tumour suppressor gene. To determine whether p73 is involved in nonastrocytic brain tumour development, we analysed 65 tumour samples including 26 oligodendrogliomas, 4 ependymomas, 5 medulloblastomas, 10 meningiomas, 2 meningeal haemangiopericytomas, 2 neurofibrosarcomas, 3 primary lymphomas, 8 schwannomas and 5 metastatic tumours to the brain, for p73 alterations. Characterization of allelic loss at 1 p36–p35 showed LOH in about 50% of cases, primarily involving oligodendroglial tumours (22 of 26 cases analysed; 85%) and meningiomas (4 of 10; 40%). PCR-SSCP and direct DNA sequencing of exons 2 to 14 of p73 revealed a missense mutation in one primary lymphoma: a G-to-A transition, with Glu291Lys change. 8 additional cases displayed no tumour-specific alterations, as 3 distinct polymorphic changes were identified: a double polymorphic change of exon 5 was found in one ependymoma and both samples derived from an oligodendroglioma, as follows: a G-to-A transition with no change in Pro 146, and a C-to-T variation with no change in Asn 204: a delG at exon 3/+12 position was identified in 4 samples corresponding to 2 oligodendrogliomas, 1 ependymoma and 1 meningioma, and a C-to-T change at exon 2/+10 position was present in a metastatic tumour. Although both LOH at 1 p36 and p73 sequence changes were evidenced in 4 cases, it is difficult to establish a causal role of the p73 variations and nonastrocytic brain tumours development.

Molecular analysis of the EGFR gene in astrocytic gliomas: mRNA expression, quantitative-PCR analysis of non-homogeneous gene amplification and DNA sequence alterations.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with tyrosine kinase activity. This report investigates the presence of mutations, amplification and/or over‐expression of the EGFR gene in 86 glial tumours including 44 glioblastomas, 21 anaplastic astrocytomas, and 21 WHO grade II astrocytomas, using polymerase chain reaction/single‐strand conformation polymorphism, semiquantitative reverse‐transcription‐polymerase chain reaction (RT‐PCR) and Southern Blot techniques. Gene amplification values were found in 34 tumours. Amplification levels were not uniform, as the transmembrane region presented lower amplification rates than extra‐ and intracellular domains. For the 19 samples with sufficient available tumour tissue we found over‐expression in 11, and no EGFR mRNA expression in three. Ten cases showed deletion transcripts, and EGFR VIII was identified in all of these cases. One of the cases with EGFR vIII also presented a truncated form, C‐958, while another showed an in frame tandem duplication of exons 18–25. We found 14 cases with sequence/structure gene alterations, including seven on which genomic novel DNA changes were identified: a missense mutation (1052C > T/Ala265Val), an insertion (InsCCC2498/Ins Pro748), three intronic changes (E6 + 72delG, E22–14C > G and E18–109T > C), a new polymorphic variant E12 + 22A > T, and one case that presented a 190 bp insertion, that was produced by the intron‐7–exon‐8 duplication and generated a truncated EGFR with intact exons 1–8 followed by an additional amino acidic sequence: Val‐Ile‐Met‐Trp. These findings corroborate that EGFR is non‐randomly involved in malignant glioma development and that different mutant forms participate in aberrant activation of tyrosine kinase pathways.

Microarray analysis of gene expression in vestibular schwannomas reveals SPP1/MET signaling pathway and androgen receptor deregulation

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (NRG1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan Low Density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.

Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity.

Identification of the 1p/19q allelic status in gliomas, primarily those with a major oligodendroglial component, has become an excellent molecular complement to tumor histology in order to identify those cases sensitive to chemotherapy. In addition to loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA) has been shown to be an alternative methodology to identify deletions of those chromosome arms. We used MLPA to explore the 1p and 19q allelic constitution in a series of 76 gliomas: 41 tumors with a major oligodendroglial component, 34 glioblastomas, and one low-grade astrocytoma. We compared the MLPA findings of the oligodendroglial cases with those previously obtained using LOH in the same samples. Thirty-eight of 41 oligodendrogliomas displayed identical findings by both LOH and MLPA, and losses at either 1p and/or 19q were identified in 12 of 35 (34%) astrocytic tumors. These findings agree with data previously reported comparing MLPA versus FISH or CGH in gliomas and suggest that MLPA can be used in the identification of the 1p/19q allelic deletions on these brain neoplasms.

Analysis of the NF2 gene in oligodendrogliomas and ependymomas

Allelic losses of chromosome 22 are commonly found in ependymomas and oligodendrogliomas, suggesting that at least one tumor suppressor gene on chromosome 22 must be inactivated during the multistep process of tumorigenesis in these glial tumors. The neurofibromatosis 2 gene (NF2) located at 22q12, is a candidate tumor suppressor gene potentially involved in the pathogenesis of gliomas. Because there have been only a few studies of the NF2 gene in glial tumors other than astrocytoma, we screened the entire 17 NF2 exons for mutations in a series of 47 nonastrocytic tumors, including 40 oligodendrogliomas and 7 ependymomas. Only one mutation was detected, a 59-base pair insertion in exon 3 from a spinal anaplastic ependymoma. These results concur with previous findings proposing preferential inactivation of the NF2 gene in a subgroup of ependymomas, and suggest that the NF2 gene is not the target of chromosome 22 aberrations in oligodendrogliomas.

Intradural Herniated Dorsal Disc: Case Report and Review of the Literature

A 67-year-old man with a 1 1/2-month history of spastic paraparesis caused by a dorsal intradural disc herniation underwent surgical treatment via a posterior approach. Dorsal herniated discs are rare, and intradural dorsal disc herniations are even more infrequent. Including this case, the medical literature reviewed describes only four such cases.